Adapting isotopic tracer and metabolic flux analysis approaches to study C1 metabolism

Single-carbon (C1, or one-carbon) substrates are promising feedstocks for sustainable biofuel and biochemical production. Crucial to the goal of engineering C1-utilizing strains for improved production is a quantitative understanding of the organization, regulation and rates of the reactions that underpin C1 metabolism. ¹³C Metabolic flux analysis (MFA) is a well-established platform for interrogating these questions with multi-carbon substrates, and uses the differential labeling of metabolites that results from feeding a substrate with position-specific incorporation of ¹³C in order to infer quantitative fluxes and pathway topology. Adapting isotopic tracer approaches to C1 metabolism, where position-specific substrate labeling is impossible, requires additional experimental considerations. Here we review recent studies that have developed isotopic tracer approaches to overcome the challenge of uniform metabolite labeling and provide quantitative insight into C1 metabolism.